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| United States Patent |
5,538,856
|
|
Levy
, et al.
|
July 23, 1996
|
Screening kit and method for diagnosing chronic immune dysfunction
syndrome
Abstract
The subject invention permits diagnosis of chronic fatigue syndrome (CFS)
by analyzing peripheral blood mononuclear cell subset populations and
activation markers. A positive diagnosis for CFS is associated with an
increase in the percentage of CD8+ cells showing CD38 or HLA-DR cell
markers or a decrease in CD11b markers in CD8+ cells. The analysis is
preferably performed using fluorochrome-labelled monoclonal antibodies
specific for a determinant of the above subset cells and activation
markers.
| Inventors:
|
Levy; Jay A. (San Francisco, CA);
Landay; Alan L. (Oak Park, IL)
|
| Assignee:
|
The Regents of the University of California (Oakland, CA)
|
| Appl. No.:
|
399774 |
| Filed:
|
March 7, 1995 |
| Current U.S. Class: |
435/7.24; 435/5; 435/975; 436/548 |
| Intern'l Class: |
G01N 033/577 |
| Field of Search: |
435/5,7.24,975
436/548
|
References Cited
U.S. Patent Documents
| 5426028 | Jun., 1995 | Levy et al. | 435/7.
|
Other References
M. Lopez et al, Biol. Abstr. 80, Abstr. No. 41755, 1985.
Holmes et al., "Chronic Fatigue Syndrome: A Working Case Definition",
Annales of Internal Medicine, 108:387-389 (1988).
Straus et al., "Persisting Illness and Fatigue in Adults with Evidence of
Epstein-Barr Virus Infection", Annales of Internal Medicine, 102:7-16
(1985).
Tobi et al., "Prolonged Atypical Illness Associated with Serological
Evidence of Persistent Epstein-Barr Virus Infection", Lancet, 1:61-64
(1982).
Krueger et al., "Antibody Prevalence to HBLV (Human Herpesvirus-6, HHV-6)
and Suggestive Pathogenicity in the General Population and in Patients
with Immune Deficiency Syndromes", Journal of Virological Methods,
21:125-131 (1988).
Wakefield et al., "Human Herpesvirus 6 and Myalgic Encephalomyelitis",
Lancet, 1:1059 (1988).
Yousef et al., "Chronic Enterovirus Infection in Patients with Postviral
Fatigue Syndrome", Lancet, 1:146 (1988).
Miller et al., "Antibody to Coxsackie B Virus in Diagnosing Postviral
Fatigue Syndrome", British Journal of Medicine, 302:140-143 (1991).
Palca, J., "Does a Retrovirus Explain Fatigue Syndrome Puzzle?", Science,
249:1240-1241 (1990).
DeFrietas et al., "Retro Viral Sequences Related to Human T-Lymphotropic
Virus Type II in Patients with Chromic Fatigue Immune Dysfunction
Syndrome", Proceedings of the National Academy of Sciences (USA),
88:2922-2926 (1991).
Kibler et al., "Immune Function in Chronic Active Epstein-Barr Virus
Infection", Journal of Clinical Immunology, 5:46-54 (1985).
Tosato et al., "Characteristic T Cell Dysfunction in Patients with Chronic
Active Epstein-Barr Virus Infection (Chronic Infectious Mononucleosis)",
Journal of Immunology, 5:3082-3088 (1985).
Caligiuri et al., "Phenotypic and Functional Deficiency of Natural Killer
Cells in Patients with Chronic Fatigue Syndrome", Journal of Immunology,
10:3306-3313 (1987).
Klimas et al., "Immunologic Abnormalities in Chronic Fatigue Syndrome",
Journal of Clinical Microbiology, 6:1403-1410 (1990).
Rouse et al., "Immunosuppression in Viral Infections", Reviews of
Infectious Diseases, 8:850-873 (1986).
Buchwald et al., "Frequency of `Chronic Active Epstein-Barr Virus
Infection` in a General Medical Practice", Journal of the American Medical
Association, 257:2303-2307 (1987).
Levy et al., "Frequent Isolation of HHV-6 from Saliva and High
Seroprevalence of the Virus in the Population", Lancet, 335:1047-1050
(1990).
Archard, "Postviral fatigue syndrome: persistance of enterovirus RNA in
muscle and elevated creatine kinase", Journal of the Royal Society for
Medicine, 81:326-9 (1988).
Lloyd et al., "Immunological Abnormalities in the Chronic Fatigue
Syndrome", Medical Journal of Australia, 151:122 (1989).
Gin et al., "Immune Function and the Chronic Fatigue Syndrome", Medical
Journal of Australia, 151:117-119 (1989).
Notkins et al., "Effect of Virus Infections on the Function of the Immune
System", Annual Review of Microbiology, 24:525 (1970).
|
Primary Examiner: Saunders; David
Attorney, Agent or Firm: Bozicevic; Karl
Fish & Richardson
Parent Case Text
CROSS-REFERENCES
This application is a divisional application of earlier filed U.S. patent
application Ser. No. 07/787,389, filed Nov. 4, 1991 issued U.S. Pat. No.
5,426,028, which is a continuation application of earlier filed
application Ser. No. 07/725,309, filed Jul. 5, 1991 (now abandoned) to
which applications we claim priority under 35 USC .sctn.120 and which
applications are incorporated herein by reference in their entirety.
Claims
What is claimed is:
1. A method for aiding in the diagnosis of chronic fatigue syndrome (CFS),
comprising the steps of:
(a) obtaining a sample of peripheral mononuclear cells from a host;
(b) contacting the sample with an immunophenotypic panel comprising
monoclonal antibodies for CD8.sup.+ antigens, CD11b antigens, CD38
antigens and HLA-DR antigens; and
(c) allowing the antibodies to bind to an antigen and classifying said host
as having CFS when said sample shows at least two of the following
conditions:
(i) a reduction in the percentage of CD8.sup.+ CD11b+ cells; or
(ii) an increase in the percentage of CD8.sup.+ CD38+ cells, or
(iii) an increase in the percentage of CD8.sup.+ HLA-DR cells as compared
to percentages of cells having said markers obtained using control hosts
not having chronic fatigue syndrome.
2. The method of claim 1, wherein binding of antibodies to said cells is
measured by detecting the presence of a fluorochrome attached to said
antibodies.
3. The method of claim 1, wherein evaluating said sample for CD11b cells
further comprises determining an increase in the number of CD11b- cells.
4. The method of claim 1, wherein said peripheral mononuclear cells are
obtained from a blood sample from said host.
5. A kit for aiding in the diagnosis of chronic fatigue syndrome,
comprising:
an immunophenotypic panel of antibodies which includes monoclonal
antibodies for CD8+ antigens, CD11b antigens, CD38 antigens and HLA-DR
antigens, wherein each of the antibodies of the panel is labelled so as to
be separately identifiable when used concurrently.
Description
TECHNICAL FIELD
The invention concerns methods for diagnosing chronic fatigue syndrome as
well as cell cultures containing an infectious agent associated with
chronic fatigue syndrome.
BACKGROUND
Chronic fatigue syndrome (CFS) is being reported with increasing frequency
in many sections of the United States as well as other parts of the world,
including England and Australia. In many patients, CFS begins with an
acute "flu-like" illness and is characterized by a debilitating fatigue
lasting for more than 3-6 months; chronic and recurrent low-grade fever,
pharyngitis, adenopathy, myalgia, arthralgia, sleep disorders and mood
disorders. A common feature is multilevel brain disorder, reflected in
mental changes such as loss of memory, vertigo, and disorientation, often
described as "spaciness." Direct measurements of brain function using, for
example, magnetic resonance imaging (MRI) have indicated abnormalities in
the central nervous system. Dementia and signs of "mental clouding" have
also been documented through psychoneurologic testing.
The infectious agent(s) for CFS is unknown; the agent is suspected to be
viral, at least in part because many viral infections are characterized by
chronic fatigue. However, post viral fatigue generally does not persist
for more than a few weeks, which is contrary to the clinical picture in
CFS. Conclusive evidence of an etiologic association of a particular known
virus to CFS has not been presented, although high levels of antibodies to
Epstein Barr virus (EBV), human herpes virus-6 (HHV-6) and the p24 core
antigen of HTLV have been reported. Further, certain immunologic
abnormalities described in CFS are often found in viral infections,
including activation of CD8+ cells. Other immunologic abnormalities
observed include decreased function of NK cells, reduced mitogenic
responses of lymphocytes and B-cell subset changes.
It is of interest to determine whether there are virologic and immunologic
parameters which are at least substantially specific for CFS so that they
can be used as a diagnostic aid in those patients who present with
symptoms including chronic fatigue. Further, definitive diagnosis of CFS
is hampered by the lack of a screening test such as an immune profile or a
serologic test to identify the etiologic agent. Identification of the
etiologic agent to produce recombinant proteins that are safe for use in
vaccines, diagnostics, and/or screening of the blood supply is greatly
desired.
Relevant Literature
The Centers for Disease Control (CDC) have attempted to define CFS in
recent cases using major and minor criteria. Holmes et al., Ann. Intern.
Med. (1988) 108:387-89. CFS has been reported to be associated with
Epstein-Barr virus. See, Strauss et al. Ann. Intern. Med. (1985) 102:7-16;
Buchwald et al. JAMA (1987) 257:2303-7; and Tobi et al., Lancet (1982)
1:61-4. High antibody levels to HHV-6 observed in some CFS patients also
do not correlate with any particular symptoms nor with virus isolation.
See, for example, Levy et al., The Lancet (1990) 335:1047-50; Krueger et
al., J. Virol. Methods (1988) 21:125; and Wakefield et al., The Lancet
(1988) 1:1059. The presence of enteroviral antigens in muscles of people
with CFS has been reported, and Coxsackie virus infections are reported to
be associated with fatigue. Archard et al., J. Royal Soc. Med. (1988)
81:326-9; and Yousef et al., Lancet (1988) 1:146-50. However, an
association of Coxsackie virus with CFS has not been confirmed. Miller,
BMJ (1991) 302:140. Likewise, the linking of an HTLV-like virus with CFS,
while suggested, has not been confirmed. Palca, Science (1990)
249:1240-41; DeFrietas et al., PNAS (May, 1991).
Decreased function of NK cells, reduced mitogenic response of lymphocytes,
B-cell subset changes, and activation of CD8+ cells with CFS have been
reported. Kibler et al., J. Clin. Immunol. (1985) 5:46-54; Tosato et al.,
J. Immunol. (1985) 134:3082-88; Murdoch Nv. Ned. J. (1988) 101:511-2;
Caligiuri et al., J. Immunol. (1987) 139:3306-13; Lloyd et al., Med. J.
Australia (1989) 151:122-24; Jin et al, Med. J. Aust. (1989) 151:117-19;
and Klimas et al., J. Clin. Micro. (1990) 28:1403-10. Infected animals and
patients suffering or recovering from a variety of acute viral infections
frequently display transient immune abnormalities and chronic fatigue.
Notkins et al., Ann. Rev. Microbiol. (1970) 24:525; Rouse and Horchov,
Rev. Insect. Dis. (1986) 8:850-73.
SUMMARY OF THE INVENTION
In accordance with the subject invention, methods are provided for
diagnosing chronic fatigue syndrome in a host presenting symptoms which
include chronic fatigue. The method involves the step of screening
peripheral blood mononuclear cells (PMC) from a host for changes in PMC
markers as compared to PMC from a healthy control. Also provided are cell
culture procedures for finding the infectious agent associated with CFS.
The cell cultures can be used as a source of genomic material for
preparing polynucleotide probes for diagnosis of CFS, as well as antigens
and vaccines for therapeutic and diagnostic applications. Propagation of
the infectious agent in vitro can be used to identify cell surface
antigens associated with the infectious agent and as a source of such
antigens.
DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows the percent expression (mean.+-.standard error) of CD11b,
CD38 and HLA-DR on CD8+ cells from normals, severely afflicted (group 1),
recovering (group 2) and all CFS patients. *p<0.05 for group 1 patients
with CFS as compared to control (Mann-Whitney U Test).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention provides methods for diagnosing CFS, together with
cell culture procedures for identifying the infectious agent associated
with CFS. The method for diagnosing CFS involves use of an immunologic
profile at least substantially specific for CFS as compared to the
immunologic profile observed with other conditions such as depression,
autoimmunity, and fatigue. To obtain the immunologic profile, peripheral
blood mononuclear cell (PMC) subset populations and markers are evaluated
for changes relative to those subset populations and markers found on PMC
obtained from a host having symptoms of chronic fatigue syndrome. The
changes associated with a diagnosis of CFS include a reduction in CD11b+
cells; an increase in CD11b- cells; and an increase in the percentage of
CD8+ cells giving CD38+ and HLA-DR+ markers. All of these individual
markers are significant (p<0.01) indicators of CFS compared to control
populations. Patients with at least 2 of the 3 abnormal markers have >90%
chance of having CFS.
The following symptoms are associated with chronic fatigue syndrome.
Previously, in order to receive a preliminary diagnosis of CSF, a patient
needed to fulfill major criteria 1 and 2 and the following minor criteria:
(1) 6 or more of the 11 symptom criteria and 2 or more of the 3 physical
criteria or (2) 8 or more of the 11 symptom criteria.
Major Criteria
1. New onset of persistent or relapsing, debilitating fatigue in a person
with no previous history of similar symptoms: fatigue that does not
resolve with bed rest and is severe enough to produce or impair average
daily activity for a period of at least 6 months.
2. Other clinical conditions that may produce similar symptoms must be
excluded by thorough evaluation, based on history, physical examination,
and laboratory findings.
Minor Criteria
Symptom criteria--began at or after onset of fatigue; must have persisted
or recurred over a period of at least 6 months.
1. Mild fever--oral temperature between 37.5.degree. and 38.6.degree. C.,
if measured by the patient--or chills.
2. Sore throat.
3. Painful lymph nodes in the anterior or posterior cervical or axillary
distribution.
4. Unexplained generalized muscle weakness.
5. Muscle discomfort or myalgia.
6. Prolonged (24 hours or greater) generalized fatigue after levels of
exercise that would have been easily tolerated in the patient's premorbid
state.
7. Generalized headaches different from ones the patient may have had in
the premorbid state.
8. Migratory arthralgia without joint swelling or redness.
9. Neuropsychologic complaints (e.g. photophobia, transient visual
scotomata, forgetfulness, excessive irritability, confusion, difficulty
thinking, inability to concentrate, depression).
10. Sleep disturbance (hypersomnia or insomnia).
11. Description of the main symptom complex as initially developing over a
few hours to a few days.
Physical Criteria
1. Low-grade fever.
2. Nonexudative pharyngitis.
3. Palpable or tender anterior or posterior cervical or axillary lymph
nodes.
*Summarized from Holmes, et al. Ann Intern Med (1988) 108:387-9.
The present invention greatly simplifies and makes more definite a
diagnosis of CFS. The method of the invention for diagnosing CFS involves
developing a peripheral white blood cell profile of the host (patient)
suspected of having CFS. The profile includes identification of the
numbers and types of cells present in a PMC sample obtained from the host
as well as an evaluation of the percentage of cells expressing activation
antigens. The profile is then analyzed to determine if any abnormal level
(which can be either an increase or a decrease, depending on the specific
marker) of a particular PMC cell subset or marker can be found in
comparison to PMC from a healthy control. The profile obtained with PMC
from a host having symptoms of CFS as compared to PMC from a host not
showing symptoms of CFS (control) is normalized by sample size or similar
criterion which will allow meaningful comparison of the profiles obtained
for different samples. Conveniently this may be accomplished by presenting
the cell population of a given type in units of cells per sample volume,
and the relative proportion of such cells as a percentage.
As is typical of diagnostic techniques, a range of differences from the
mean value of any specific component of the profile is to be expected.
Patents with mild symptoms are expected to exhibit only slight differences
for any of the individual parameters while patents with severe symptoms
are expected to exhibit greater differences. However, as with any
diagnostic assay, different results can occur in patients who do not
respond in the normal fashion of the general population, so that slight
differences can occur in patients with severe symptoms and vice versa. Any
of the normal statistical evaluation methods used to determine the
significance of a single value from a mean can be used to evaluate the
significance of a given difference. For example, values that fall outside
one standard deviation from the mean have about a 90% chance of being
statistically different from the mean. Since a range of mean.+-.one
standard deviation would still classify as abnormal relatively large
portion of the general population, values more than two, preferably at
least three, more preferably at least four, standard deviations from the
mean are generally used to evaluate whether a single value (such as
percentage of CD8+ cells) is outside the normal range and thus indicative
of abnormality (i.e., CFS). However, it should be recognized that the
present invention involves testing for the indicated results and any
statistically significant difference and not necessarily finding such
differences, since it is probable that a large number of individuals test
for CFS will be found not to have CFS.
The subset cell profile can be obtained by any of a variety of methods
including flow cytometry (FACS); see, for example, Levy et al., Clin.
Immunol. Immunopathol. (1985) 35:328. In the FACS method of analysis,
monoclonal antibodies to a variety of subset cells bind to and identify
phenotypic antigens present on immune system cells. Commercially available
antibodies exist that can detect the presence of these markers, so that
preparation of the antibodies is not required. One supplier of these (and
other) antibodies is Becton Dickinson Immunocytometry Systems of Mountain
View, Calif. Other antibodies which identify the same or a closely linked
antigenic marker would be expected to give similar diagnostic results.
Thus, where a marker antigen is designated in the specification or claims
by reference to a particular monoclonal antibody with which it binds
(e.g., CD11b, CD38), such a designation will be understood to encompass
that marker even if different monoclonal antibodies are used in the
identification. Phenotypic markers of interest include general markers for
various subset cell types including CD3 for total T cells, CD4 for T
helper/inducer cells, CD8 for T suppressor/cytotoxic cells, and CD16/56
for NK cells; CD8-expressing subset markers such as CD11b for T suppressor
cells, CD38 for activated T suppressor/cytotoxic cells, HLA-DR for
activated T suppressor/cytotoxic cells, and CD57; and CD4 expressing
markers such as CD25 and HLA-DR for activated T helper/inducer cells.
Of particular interest in diagnosing CFS is the evaluation of CD8+ cells,
particularly for a percentage increase in the subsets of cells exhibiting
activation antigens CD38 and HLA-DR as well as for a decrease in the
percentage of CD11b+ cells (or a corresponding increase in CD11b- cells).
While it is anticipated that further studies using additional monoclonal
antibodies against other lymphocyte antigens may reveal additional
markers, such additional markers (if found) will not detract from the
present assay. Thus a diagnosis of CFS can be made by evaluating PMC cells
for the markers discussed in this paragraph and determining whether one or
more statistically different values exist in a given patient (compared to
the corresponding values for the general population). A definitive
diagnosis is made by determining a statistical difference in at least two
of the indicated three values.
The invention also contemplates a kit for diagnosis of chronic fatigue
syndrome comprising a plurality of component monoclonal antibodies or
other specific binding molecules, where each of the component antibodies
is specific for one of the cell markers CD11b, CD38, or HLA-DR. The
antibodies are packaged together in a single container or in a plurality
of individual containers for ease of use in the diagnostic assay described
above. The antibodies can be bound to a solid support or be provided in a
form suitable for preparation of antibody-containing solutions. The kit
will normally also contain an antibody specific for the CD8 antigen.
Preparation of diagnostic kits for the determination of specific antibody
binding is well known in the art and need not be described here in detail.
The novelty of this aspect of the invention lies not in preparation per se
of the kit but in the selection of the specific antibodies. Prior to the
present invention there has been no purpose for packaging together the
antibodies or other binding molecules of the specificities described here.
Normal detectable labels can be used for the individual antibody
components, such as fluorochromes. When a kit of the invention is designed
for concurrent use of the antibodies, as occurs in fluorescent cell
sorting, each of the component antibodies is labelled so as to be
separately identifiable (e.g., with a fluorochrome that fluoresces at a
different wavelength).
Methods for identifying and subsequently propagating relatively large
amounts of the causative agent include in vitro tissue culture assays
using cells susceptible to infection by the agent are also provided by the
invention. Cultures comprising the infectious agent may be obtained by
co-cultivating a human or animal cell culture or cell line with PMC of a
CFS patient. Generally the source of infectious agent is a fluid which has
been in contact with a cell from a host having been preliminarily
diagnosed as having CFS, which fluid is capable of inducing CFS-associated
changes such as in markers or subset populations in cultured, normal PMC
cells. The cultured cells find use as a source of infectious agent
(isolate) which may be used directly as a source of genomic material for
preparing probes for diagnosis, antigens and vaccines, and for
post-therapeutic and diagnostic purposes. Agent-specific cells which can
be used to culture the CSF agent include PMC cells and selected subsets,
particularly NK cells, macrophages, B cells and CD8+ cells. Various cell
lines can also be used, including human cell lines such as a T cell line
(for example SUP-T, MT-4), monocyte cell lines (for example U937), B cell
lines (for example RAMOS, Raji), fibroblastoid cells (HOS for example),
human skin fibroblasts, and neuroblastoma cells (for example SK-N-Mc).
Animal cell lines which find use include 3T3 (mouse), DHK (hamster),
chicken embyro, mink lung, bat lung, NRK (rat kidney), and VERO (monkey).
The general procedures for infecting and culturing the cells with the
infectious agent are as follows. A cultured cell or cell line is placed in
contact with a source of agent which is capable of inducing CSF associated
changes in cultured PMC cells, particularly increasing the percentage of
CD8+ cells expressing CD38+ or HLA-DR+. The source can be a fluid obtained
directly from a host having symptoms of CFS, such as a bodily fluid,
particularly whole blood, serum, saliva, and cerebrospinal fluid. Bodily
fluid from an animal inoculated with any of the foregoing materials, as
well as culture fluids (conditioned medium) from for example, cultures of
PMC cells obtained from a CSF host may also be used.
Viral infection of the cells is followed by monitoring for a virus-related
cell change over time. Virus infection generally is characterized by the
appearance of virus-specific antigens, so the viral infection is properly
followed by immunological methods known to those skilled in the art (for
example, IFA) for detecting antigens. In addition, detection may be
accomplished by microscopic observation of cytopathological changes in the
inoculated cells, such as the cell ballooning and degeneration observed in
RAMOS B cells after culture with PMC supernatant from CFS patients.
After viral infection and propagation, the virus can be isolated, if
desired, by conventional means for releasing and purifying virus particles
from cells. For example, virus particles may be isolated by lysing the
cells and subjecting the lysate to standard techniques for fractionating
samples containing viruses. Where possible, techniques that might disrupt
virus particles should be avoided. The isolated particles will reproduce
the virus-related cell change when cells are exposed to virus particles.
It may be desirable for a variety of reasons to further purify the
particles present in a sample containing particles of the invention. For
example, if a virus particle is to be treated and employed as a vaccine or
in an immunoassay, there ordinarily should be as little in the way of
extraneous protein contamination as possible. Thus, the particle should be
substantially free of host proteins.
CFS viral antigens may be obtained from a variety of sources. The antigen
may be present on an intact virus particle, a partially degraded virus
particle, a protein- or carbohydrate-containing molecule in solution, or
any other physical form, including an antigen that has been combined
either chemically or physically with a particle or solid surface, such as
by attaching antigens to the surface of a test tube or to suspended
particles, such as red blood cells or latex particles. An antigen of the
invention is defined as a substance containing at least one epitopic site
of a virus particle.
To obtain CFS viral antigens, the antigens, whether soluble or in some
other form, are typically first separated from cellular contaminants, such
as animal cells, cell debris, and cellular microorganisms such as
bacteria. This gross separation is generally accomplished by
centrifugation or by filtration using standard techniques. Ordinary
filters having an average pore diameter of 0.45.mu. are useful in
retaining gross contamination and passing through the antigens.
Additionally, antigens of the invention may be separated from undesired
water-soluble materials after gross contamination is removed. Where it is
desired to recover either intact virus particles or their water-insoluble
fragments, it is convenient to simply remove all water soluble
constituents from the sample. Suitable techniques include ultrafiltration
through a membrane, use of selective flocculating or protein-precipitating
agents (such as polyethylene glycol and ammonite sulfate), and
chromatography. Chromatography is the most versatile method since it can
be readily scaled up for commercial manufacture of antigen. Gel
chromatography systems using cross-linked dextran beads are typical of the
materials used. A column of a suitable gel can be selected which will
permit diffusion of proteins and low molecular weight substances into the
void volume of the gel beads, thereby retarding the progress of these
contaminants through the column, while allowing whole virus particles to
pass through virtually unimpeded. When a particular antigen is desired,
other gel sizes can be selected to provide for isolation of an antigen of
any particular size. The gel which is selected will thus be a matter of
routine experimentation.
Any of the techniques described herein can be combined as desired. For
example, isolation of particles on a cesium chloride or sucrose density
gradient can be followed by disruption of particles using any of a variety
of techniques and isolation of a viral antigen on gel electrophoresis,
selecting for proteins binding to antibodies, e.g., antisera, specific for
CFS antigens.
One technique that is particularly suitable for isolating soluble protein
antigens or particle fragments is affinity chromatography. Antibodies
capable of binding antigens of the invention are covalently linked or
adsorbed to an insoluble support using conventional procedures. The
insolubilized antibody is laced in a column. A sample containing antigen
is passed through the column, where it binds to the insolubilized
antibody. The immunologically-bound antigen is washed with buffer and can
then be released by, for example, changing the ionic strength or pH of the
wash buffer. Generally, an acidic pH is effective for releasing the bound
antigen. The technique is highly effective in separating closely related
proteins from the antigens of the invention.
Antigens of the invention can be used as a vaccine. A preferred starting
material for preparation of a vaccine is the particle antigens produced by
tissue culture of the infectious virus. The antigens are preferably
initially recovered as intact particles as described above. However, it is
also possible to prepare a suitable vaccine from particles isolated from
other sources or non-particle recombinant antigens. When non-particle
antigens are used (typically soluble antigens), proteins native to the
viral envelope are preferred for use in preparing vaccines. These proteins
can be purified by affinity chromatography, also described above.
If the purified protein is not immunogenic per se, it can be bound to a
carrier to make the protein immunogenic. Carriers include bovine serum
albumin, keyhole limpet cyanin and the like. It is desirable, but not
necessary to purify antigens to be substantially free of human protein.
However, it is more important that the antigens be free of proteins,
viruses, and other substances not of human origin that may have been
introduced by way of, or contamination of, the nutrient medium, cell
lines, tissues, or pathological fluids from which the virus is cultured or
obtained.
Vaccination can be conducted in conventional fashion. For example, the
antigen, whether a viral particle or a protein, can be used in a suitable
diluent such as water, saline, buffered salines, complete or incomplete
adjuvants, and the like. The immunogen is administered using standard
techniques for antibody induction, such as by subcutaneous administration
of physiologically compatible sterile solutions containing inactivated or
attenuated virus particles or antigens. An immune response producing
amount of virus particles is typically administered per vaccinizing
injection, usually in a volume of one milliliter or less.
In addition to being used in vaccines, the compositions can be used to
prepare antibodies to CFS virus particles. The antibodies can be utilized
directly as antiviral agents. To prepare antibodies, a host animal is
immunized using the virus particles or, as appropriate, non-particle
antigens native to the virus particle are bound to a carrier as described
above for vaccines. The host serum or plasma is collected following an
appropriate time interval to provide a composition comprising antibodies
reactive with the virus particle. The gamma globulin fraction or the IgG
antibodies can be obtained, for example, by use of saturated ammonium
sulfate or DEAE Sephadex, or other techniques known to those skilled in
the art. The antibodies are substantially free of many of the adverse side
effects which may be associated with other anti-viral agents such as
drugs.
The antibody compositions can be made even more compatible with the host
system by minimizing potential adverse immune system responses. This is
accomplished by removing all or a portion of the Fc portion of a foreign
species antibody or using an antibody of the same species as the host
animal, for example, the use of antibodies from human/human hybridomas
(see below).
The antibodies can also be used as a means of enhancing the immune response
since antibody-virus complexes are recognized by macrophages. The
antibodies can be administered in amounts similar to those used for other
therapeutic administrations of antibody. For example, pooled gamma
globulin is administered at 0.02-0.1 ml/lb body weight during the early
incubation of other viral diseases such as rabies, measles and hepatitis B
to interfere with viral entry into cells. Thus, antibodies reactive with
the CFS virus particle can be passively administered alone or in
conjunction with another anti-viral agent to a host infected with a CFS
virus to enhance the immune response and/or the effectiveness of an
antiviral drug.
Alternatively, anti-CFS-virus antibodies can be induced by administering
anti-idiotype antibodies as immunogen. Conveniently, a purified
anti-CFS-virus antibody preparation prepared as described above is used to
induce anti-idiotype antibody in a host animal. The composition is
administered to the host animal in a suitable diluent. Following
administration, usually repeated administration, the host produces
antiidiotype antibody. To eliminate an immunogenic response to the Fc
region, antibodies produced by the same species as the host animal can be
used or the Fc region of the administered antibodies can be removed.
Following induction of anti-idiotype antibody in the host animal, serum or
plasma is removed to provide an antibody composition. The composition can
be purified as described above for anti-CFS-virus antibodies, or by
affinity chromatography using anti-CFS-virus antibodies bound to the
affinity matrix. The antiidiotype antibodies produced are specific for
CFS-virus particles.
When used as a means of inducing anti-CFS-virus antibodies in a patient,
the manner of injecting the antibody is the same as for vaccination
purposes, namely intramuscularly, intraperitoneally, subcutaneously or the
like in an effective concentration in a physiologically suitable diluent
with or without adjuvant. One or more booster injections may be desirable.
The induction of anti-CFS-virus antibodies can alleviate problems which
may be caused by passive administration of anti-CFS-virus antibodies, such
as an adverse immune response, and those associated with administration of
blood products, such as infection.
In addition to therapeutic uses, the particles and antigens of the
invention, as well as the genetic material, can be used in diagnostic
assays. Methods for detecting the presence of CFS comprise analyzing a
biological sample such as a blood or CFS sample for the presence of an
analyte associated with CFS virus. The analyte can be a nucleotide
sequence which hybridizes with a probe comprising a sequence of at least
about 16 consecutive nucleotides, usually 30 to 200 nucleotides, up to
substantially the full sequence of a cDNA sequence. The analyte can be RNA
or cDNA.
The analyte can be a virus particle having at least one of the following
characteristics: obtainable from cells susceptible to infection with CFS,
including cells from CFS patients; capable of inducing expression of
virus-specific surface antigen in a cell susceptible to infection by the
particle, the surface antigen being recognized by serum from a host
infected with CFS and not by serum from a non-infected host; and capable
of inducing cytopathological changes in exposed immune cells, such as
ballooning and degeneration of RAMOS cells. The analyte can comprise an
antibody which recognizes an antigen, such as a cell surface viral antigen
or a CFS virus particle. The analyte can also be a CFS viral antigen.
In order to detect an analyte, where the analyte hybridizes to a probe the
probe may contain a detectable label. Likewise, where the analyte is an
antibody or an antigen, either a labelled antigen or antibody,
respectively, can be used to bind to the analyte to form an immunological
complex, which can then be detected by means of the label.
Typically, methods for detecting analytes such as surface antigens and/or
whole particles are based on immunoassays. Immunoassays can be conducted
either to determine the presence of antibodies in the host that have
arisen from infection by CFS virus or by assays that directly determine
the presence of virus particles or antigens. Such techniques are well
known and need not be described here in detail. Examples include both
heterogeneous and homogeneous immunoassay techniques and are based on the
formation of an immunological complex between the virus particle or its
antigen and a corresponding specific antibody. Heterogeneous assays for
viral antigens typically use a specific monoclonal or polyclonal antibody
bound to a solid surface. Sandwich assays are becoming increasingly
popular. Homogeneous assays, which are carried out in solution without the
presence of a solid phase, can also be used, for example by determining
the difference in enzyme activity brought on by binding of free antibody
to an enzyme-antigen conjugate.
When assaying for the presence of antibodies induced by CFS viruses, the
viruses and antigens of the invention can be used as specific binding
agents to detect either IgG or IgM antibodies. Since IgM antibodies are
typically the first antibodies that appear during the course of an
infection, when Ig synthesis may not yet have been initiated, specifically
distinguishing between IgM and IgG antibodies present in the blood stream
of a host will enable a physician or other investigator to determine
whether the infection is recent or chronic. This information can then be
used in conjunction with the immunological profile, particularly where the
immunological profile is abnormal but not significantly different from
normal. For example, if only IgM antibodies are present, it may be useful
to reevaluate the host at 3-month intervals.
The genetic material of the invention can itself be used in numerous assays
as probes for genetic material present in naturally occurring infections.
It may be amplified as necessary. One method for amplification of target
nucleic acids, for later analysis by hybridization assays, is known as the
polymerase chain reaction or PCR technique. The PCR technique can be
applied to detecting virus particles of the invention in suspected
pathological samples using oligonucleotide primers. The PCR method is
described in a number of publications, including Saiki et al., Science
(1985) 230:1350-1354; Saiki et al., Nature (1986) 324:163-166; and Scharf
et al., Science (1986) 233:1076-1078. Also see U.S. Pat. Nos. 4,683,194;
4,683,195; and 4,683,202.
For both in vivo use of antibodies to CFS-virus particles and proteins and
anti-idiotype antibodies and diagnostic use, it may be preferable to use
monoclonal antibodies. Monoclonal anti-virus particles antibodies or
anti-idiotype antibodies can be produced as follows. The spleen or
lymphocytes from an immunized animal are removed and immortalized or used
to prepare hybridomas by methods known to those skilled in the art. To
produce a human-human hybridoma, a human lymphocyte donor is selected. A
donor known to be infected with a CFS virus (where infection has been
shown for example by the presence of anti-virus antibodies in the blood or
by virus culture) may serve as a suitable lymphocyte donor. Lymphocytes
can be isolated from a peripheral blood sample or spleen cells may be used
if the donor is subject to splenectomy. Epstein-Barr virus (EBV) can be
used to immortalize human lymphocytes or a human fusion partner can be
used to produce human-human hybridomas. Primary in vitro immunization with
peptides can also be used in the generation of human monoclonal
antibodies.
Antibodies secreted by the immortalized cells are screened to determine the
clones that secrete antibodies of the desired specificity. For monoclonal
anti-virus particle antibodies, the antibodies must bind to CFS virus
particles. For monoclonal antiidiotype antibodies, the antibodies must
bind to anti-virus particle antibodies. Cells producing antibodies of the
desired specificity are selected.
The following examples are offered by way of illustration and not by
limitation.
EXAMPLE 1
Immunologic Profile of Patients With CFS Subjects
A total of 147 consecutive patients (30 males and 117 females, age range
15-80, median age 38) who were presented for evaluation as having CFS were
studied from September 1989 to November 1990. As determined either at the
time of evaluation or by past medical history, their illness had been
present for 1-3 years. All patients met the criteria for CFS as defined by
the CDC (Kruesi M., J. Clin. Psychiatry (1989), 50(2):53-6) and in Table I
below.
TABLE I
______________________________________
Symptoms of Patients with CFS
Group 1.sup.b
Group 2.sup.c
Total CFS Patients
Symptoms (n = 67) (n = 21) (n = 47)
______________________________________
Exhaustion/Fatigue
100.sup.a
5 70
Post exertional weak-
100 24 92
ness
Muscle Weakness
96 0 56
Severe Cognitive
92 0 74
Dysfunction.sup.d
Abdominal/Gastro-
90 0 84
intestinal Distress
Nausea 89 0 46
Neuroirritability
89 5 69
Sleep Disorder.sup.e
89 0 58
Twitching/Myoclonus
89 10 56
Frequent Headache
81 14 59
Balance problems
75 5 66
Depression.sup.f
73 0 46
Chills 55 0 33
Sore throat 33 5 20
Lymph Node Pain
30 0 19
______________________________________
.sup.a Figures give percent of patients exhibiting symptoms.
.sup.b Patients in this group have 25% or less normal physical activity
and major CFS symptoms.
.sup.c Patients in this group have 75% or more normal physical activity
and mild CFS symptoms. They had marked improvement for at least 2 months
in their clinical condition at the time of study.
.sup.d Short term memory loss, encoding, stimulus recognition.
.sup.e Hypersomnia and hyposomnia.
.sup.f In the majority of cases, the onset of depression occurred 6 month
after the onset of the illness.
Control subjects consisted of healthy individuals (n=50) living in San
Francisco and working at the UCSF Medical Center and subjects (n=30)
seeking medical attention for clinical conditions other than CFS. The
majority of these other individuals were being seen for routine physical
examinations. Immunophenotypic data on these two control groups were not
significantly different, so they were combined for the study as one
control group (30 males, 50 females, age range 20-55, median age 32).
Additional control populations evaluated and spouses and family members of
CSF patients (n=11); medical personnel in contact with CFS patients
(n=11); patients with acute viral-like illness (n=15); patients with
systemic lupus erythematosus (SLE) (n=12) as defined by the American
Rheumatology Association; patients with documented depression (n=10); and
individuals with prolonged fatigue without any other clinical criteria for
CFS (n=6). All non-CFS subjects were referred by physicians in the San
Francisco and Chicago areas.
Blood Samples
EDTA anticoagulated blood was collected for flow cytometric studies, white
blood cell counts, and differential counts. An additional serum sample was
obtained for viral serologic studies.
Viral Serology
Quantitative serology was done to measure the levels of antibodies to
various viruses in randomly selected sera from 40 healthy laboratory
personnel controls compared to those obtained with 63 CFS patients
representing 23 individuals in group 1 and 27 individuals in group 2 (as
defined in Table I). The viral agents evaluated included those proposed as
"candidate" agents for CFS as well as other viruses. Antibody to
Adenovirus, Coxsackie B4, human herpes virus 6 (HHV6), human T cell
leukemia virus I/II (HTLV-I/II), human immunodeficiency virus (HIV),
rubeola, and papovavirus were assayed by indirect immunofluorescence
(Lennette, J. Clin Microbiol. (1987) 25:199-200); antibody response to
cytomegalovirus was evaluated by immune adherence hemagglutination (CMV)
Lennette, J. Clin. Microbiol. (1978) 7:282-85); and Epstein-Barr virus
(EBV) early antigen (EA), viral capsid antigen (VCA) and nuclear antigen
(EBNA) were measured by both indirect immunofluorescence and
anti-complement indirect immunofluorescence (Reedman, Int. J. Cancer
(1973) 11:499-520; and Lennette (1987) supra).
Sample Preparation and Flow Cytometric Analysis
Lymphocyte and monocyte populations were analyzed by flow cytometry using
dual color direct immunofluorescence after whole blood lysis (Coon et al.,
Lab. Invest (1987) 57:453-79). The panels of fluorescein isothiocyanate
(FITC) or phycoerythrin (PE) monoclonal antibodies used are listed in
Table II below. A single laser flow cytometer (FACScan.TM.): Becton
Dickinson Immunocytometry Systems, San Jose, Calif.) which has the
capability of discriminating forward and side scatter as well as two
fluorochromes was used with the Consort 30 software.
TABLE II
______________________________________
Immunophenotyping Panel Used for Evaluation of CFS Patients
Cell Subset Antibody Specificity
______________________________________
CD3 Total T
CD4 T helper/inducer (T H/I)
CD3CD8 Cd8.sup.+ T suppressor/cytotoxic (T S/C)
CD8CD11b Suppressor T cell
CD8CD28 Cytotoxic T cell
CDUCD57 T S/C Subset
CD8Leu8 T S/C Subset
CD16CD56 Natural killer
CD20 B cell
CD5 CD20 B cell subset
CD14 Monocyte
Activation Markers
CD4CD25 Activated T H/I
CD4HLA-DR Activated T H/I
CD8CD38 Activated T S/C
CD8HLA-DR Activated T S/C
CD8CD25 Activated T S/C
CD16CD25 Activated NK
CD16HLA-DR Activated NK
CD14CD25 Activated NK
Cell Adhesion Markers
CD11a Adhesion molecule integrin family
(LFA1-a)
CD18 Adhesion molecule integrin family
9LFA1-b)
CD44 Homing receptor
CD54 Intracellular adhesion molecule
(ICAM-1)
______________________________________
The method used was as follows. A 100 .mu.l sample of whole blood was
aliquoted into tubes, monoclonal antibody was added, and the mixture
incubated for 15 minutes at room temperature. Red cells were lysed with a
commercial lysing reagent (FACsLyse.TM.) for 10 minutes, and the lysate
spun down and washed with phosphate buffered saline (PBS) containing 3%
fetal calf serum and 0.1% sodium azide. The white cells were resuspended
in the wash buffer and fixed with a final concentration of 1%
paraformaldehyde. Lymphocyte gates were confirmed by the HLE FITC (CD45),
LeuM3 PE (CD14) antibody combination (Leukegate). Evaluation of the cell
adhesion antigens on lymphocyte, monocyte, and neutrophil populations was
performed using indirect immunofluorescence staining.
Data Analysis and Statistics
The Mann-Whitney U and Kruskall-Wallis tests were used to evaluate
difference in immunologic markers between the groups of patients, since
these markers are known to have non-Gaussian distribution (Siegel, (1956)
McGraw Hill Book Co., New York). Student's T-test for independent samples
or one-way analysis of variance (SPSS-PC.sup.+ Release 4.0, SPSS, Inc.,
Chicago) was used to evaluate differences between the groups for other
continuous variables. When more than 2 groups were evaluated, the Scheffe'
test was employed to identify the groups for which the differences were
significant.
Clinical Evaluation of CFS Patients
The individuals referred with the suggested diagnosis of CFS were evaluated
for a large number of symptoms (Table I), past medical history and
physical findings. A full report on the clinical aspects of their illness
will be the subject of another publication. All the patients had a past
history of fatigue for more than six months, post exertional weakness
substantially worse than previously observed in the past, muscle weakness,
myalgias and frequent headaches. Many had gastrointestinal symptoms with
nausea, and neurologic findings that include sleep disorders, severe
cognitive dysfunction, neuroirritability, myoclonus and balance problems
(Table I). About 50% gave a medical history of flu-like illness at the
onset of this clinical condition. Only 8 patients had depression prior to
the onset of the illness, but many (80%) developed depression after 2
years of illness. Based on their symptomatology, the CFS patients could be
placed into three groups: Group 1 consisted of 67 patients whose illness
was so severe that they had less than 25% of their normal daily activity
and multiple symptoms, Group 2 consisted of 21 patients who initially had
severe symptoms and incapacitating illness like Group 1; however, at the
time of evaluation they had made substantial improvement for at least 2
months and regained 75% or ore of their normal physical activity with only
mild symptoms; 59 individuals with reduced physical activity continued to
have moderate symptoms. Healthy controls had none of the symptoms listed
in Table I on a persistent basis.
Viral Serology
The prevalence and titers of antibodies to CMV, EBV-VCA, EBNA, rubeola,
adenovirus type 2, and the papovavirus (BK virus) did not differ between
healthy controls and CFS patients (Table III).
TABLE III
______________________________________
Comparative Serosurvey of Antiviral Antibodies
in CFS Patients and Control Subjects
Healthy Controls
CFS Patients
N = 40 N = 63
Virus GMT.sup.a
(%+) GMT (%+)
______________________________________
Cytomegalovirus
42 (40) 67 (46)
Epstein-Barr
VCA 314 (95) 393 (95)
EA 25 (15) 40 .sup. (51).sup.b
EBNA 206 (95) 96 (95)
Rubeola 473 (98) 577 (89)
HHV-6 104 (98) 201 (100)
Adenovirus 285 ((%) 155 (81)
Coxsackie B4 113 (65) 134 .sup. (90).sup.b
Papovavirus BK
83 (55) 61 (44)
HTLV-II (0) (0)
HIV-1/HIV-2 (0) (0)
______________________________________
.sup.a GMT = geometric mean titer.
.sup.b The seroprevalence is statistically significant (p<0.001) by Chi
square analysis.
Evaluation of CFS patients based on clinical status as defined in Table I
also showed no differences in these viral antibody titers as compared to
controls. No antibodies to HTLV-I/II and HIV were found in any subjects.
Antibody titers to HHV-6 in the CFS patients were twice as high as those
found in controls, but were not significantly different (p>0.05). The
prevalence of antibodies to Coxsackie B4 virus was significantly higher in
the CFS group compared to controls (90% vs. 65%) (p<0.001), but the
geometric mean titers were approximately the same. Moreover the prevalence
of antibodies to EBV-EA was significantly different between the CFS
patients and controls 51% vs. 15%) (p<0.001). This finding was specific
for EA since the VCA and EBNA seropositivity rates for both groups were
identical.
Peripheral white Blood Cell Analyses: Lymphocyte nd Monocyte Phenotypic
Profile
The total number of white blood cells (6-10.times.10.sup.3 /mm.sup.3) was
not different between CFS patients and healthy control groups. Evaluation
of the total lymphocyte, monocyte and neutrophil populations also showed
no differences between control and CFS patients whether the latter were
considered as total or separate groups (Table I). Cell surface phenotypic
analysis of the major lymphocyte populations including T (CD3), B (CD20),
and NK (CD16+/CD56+) cells indicated no significant differences between
CFS patients and controls (Table IV).
TABLE IV
______________________________________
Peripheral Blood Phenotypic Profile in CFS
and Healthy Individuals
CFS Controls
Surface Markers (n = 147) (n = 80)
______________________________________
% CD3.sup.+ (total T cell)
71 .+-. 8.sup.a
74 .+-. 8
% CD4.sup.+ cell 46 .+-. 9 48 .+-. 9
(T helper/inducer cell)
CD4.sup.+ cell number
834 .+-. 315
889 .+-. 273
% CD8.sup.+ cell 24 .+-. 8 22 .+-. 4
(T suppressor/cytotoxic)
CD8.sup.+ cell number
468 .+-. 227
488 .+-. 220
% CD20.sup.+ cell (B cells)
11 .+-. 16
10 .+-. 3
% CD16/CD56.sup.+ (NK cell)
13 .+-. 4 14 .+-. 8
Helper/Suppressor Ratio
1.7 .+-. 0.5
1.8 .+-. 0.4
% CD4+ Cells Expressing
CD25 33 .+-. 7 34 .+-. 6
HLA-DR 12 .+-. 6 10 .+-. 7
% CD16+ Cells Expressing
CD25 4 .+-. 1 3 .+-. 2
HLA-DR 3 .+-. 1 3 .+-. 1
% CD14+ Cells Expressing
CD25 >1 >1
______________________________________
.sup.a Data represent mean .+-. standard deviation.
Evaluation of the percentage and absolute number of CD4.sup.+ and CD8.sup.+
T cells also demonstrated no significant differences (p>0.05) from the
controls (Table IV). Analysis of the CD4:CD8 ratios of cells in our
patient population showed 88% had ratios within our normal range (1-2.5),
5% had decreased CD8.sup.+ cells and 7 had increased CD8.sup.+ cells
(ratio<1.0). Taken together the average CD4/CD8 ratio for all CFS patients
(1.7+0.5) did not differ significantly from controls (1.8+0.4) (p>0.05).
Analysis of activation antigens (CD25 and HLA-DR) on CD4.sup.+ T cells, NK
cells or monocytes showed no significant differences in CFS patients from
healthy controls. Analysis of a B cell subset (CD5/CD20) that has been
found to be elevated in autoimmune disease (Casali et al., Science (1987),
236:77-81) also indicated no differences between CFS and healthy controls.
In addition, flow cytometric studies of the cell adhesion antigens (CD11a,
CD18, CD44, CD54) showed no differences in percentage positive or the
fluorescence intensity of cells obtained from CFS patients compared to
healthy controls.
CD8 Cell Subset Analysis
Previous cell surface marker studies in documented acute viral infections
such as EBV, CMV, and HIV infection, have shown elevation of CD8.sup.+
cells which express activation antigens (CD38, HLA-DR) (Carney et al., J.
Immunol., 1981, 126:2114-16; Tomkinson et al., J. Immunol., 1987,
139:3802-07 and Landay et al., Sixth International Conference on AIDS, San
Francisco, Calif., 1990, p. 141). In the case of the herpes viruses, these
cell numbers return to normal 2 to 4 weeks following infection. We
evaluated a number of cell surface antigens expressed on CD8.sup.+ T cells
from the 147 CFS patients and compared them to the 80 healthy controls
(FIG. 1). In addition, the results with the two separate clinical
subgroups (Table I) were considered.
Three markers gave noteworthy results. With the total CFS patients, the
population of CD8.sup.+ cells expressing CD11b were somewhat decreased
when compared to the normal controls (19.+-.16 vs. 25.+-.10) indicating a
drop in the phenotypic suppressor CD8.sup.+ T cell compartment. Since the
number of total CD8.sup.+ cells is not changed, a concomitant increase in
the phenotypic cytotoxic (CD8.sup.+ CD28.sup.+ CD11b-) population is seen.
This result was confirmed in a preliminary study showing a rise in the
CD8.sup.+ CD28.sup.+ population in these patients. Evaluation of
activation antigens on the CD8.sup.+ cells showed an increase in CD38
(47.+-.20 vs 35.+-.12 for Controls) and HLA-DR (22.+-.8 vs 14.+-.6 for
controls) expression. An additional activation antigen CD25, as well as
the CD57 and Leu8 antigens were expressed on the CD8.sup.+ cells at a
level comparable to controls.
When these same CD8.sup.+ cell subsets were considered in Group 1 patients,
the differences as compared to healthy controls (FIG. 1) reached
statistical significance. In contrast, evaluation of the CD8.sup.+ cell
subsets among the CFS patients who improved in their clinical status
(Group 2) showed no significant differences from healthy controls (FIG.
1). The 59 patients who had moderate symptoms also had CD8.sup.+ cell
abnormalities, but they were not statistically significant. Further
evaluation of Group 2 showed only 10% of patients had two or more
significantly abnormal results among the CD8.sup.+ CD11b.sup.-, CD8.sup.+
CD38.sup.+, or CD8 +HLADR.sup.+ subsets, whereas among the Group 1
patients 85% had 2 or more abnormal results. These data indicate a high
probability (90%) of having active CFS if an individual has two or more of
the CD8.sup.+ cell subset alterations.
To control for possible alteration in cell surface antigens by the whole
blood lysis procedure, a subset of patients was evaluated following
Ficoll-Hypaque isolation of peripheral blood mononuclear cells. No
differences were seen between the whole blood lysis procedure and
Ficoll-Hypaque purified cells.
CD8 Cell Markers in Other Control Populations
To evaluate the specificity of the CD8.sup.+ cell alteration in CFS
patients, several individuals with other clinical conditions were
evaluated with the same panel of monoclonal reagents. Among patients who
had an acute viral-like illness (common cold or flu-like illness), a
number of observations were made. The initial cellular response
demonstrated an increase in both percentage and absolute numbers of
natural killer cells (CD16.sup.+ /CD56.sup.+). These natural killer cells
were CD8.sup.+, CD38.sup.+, CD11b.sup.+, and HLADR.sup.- as determined by
multiparameter flow cytometric studies. In several of the individuals
studied we noted this initial natural killer response was followed by an
increase in activated CD8/CD11b T cells (CD38.sup.+ HLA-DR+). In all these
subjects, recovery 1-2 weeks later was accompanied by a return to normal
of all these immunologic parameters. All other CD8.sup.+ cell markers were
normal in these individuals.
Evaluation of CD8.sup.+ subsets in patients with a diagnosis of depression
showed no significant differences compared to healthy controls.
Furthermore, family members and contacts of CFS patients demonstrated
normal CD8.sup.+ subsets as well as those individuals presenting with
fatigue other than CFS (Table V).
TABLE V
__________________________________________________________________________
CD8.sup.+ Cell Surface Markers in Control Populations
Chronic
Acute
Autoimmune
Family Fatigue
Autoimmune
Healthy
CD8.sup.+ Cells
Viral
Depression
Members.sup.b
Contacts.sup.c
CF Alone
Disease
Controls
Expressing
(n = 15)
(n = 10)
(n = 11)
(n = 11)
(n - 6)
(n = 12)
(n - 80)
__________________________________________________________________________
CD11b 42; 12
28; 10
27; 12
27; 8
30; 12
28; 6 25; 10
CD38 .sup. 63; 10.sup.a
26; 8 28; 7 32; 6
25; 6 45; 12
53; 12
HLA-DR 12; 3
10; 5 10; 6 12; 6
9; 3 15; 4 14; 6
CD16/56
24; 7
12; 4 8; 2 11; 4
10; 5 12; 6 14; 8
__________________________________________________________________________
.sup.a Statistically significant (Schiffe test) compared to healthy
controls at p<0.05.
.sup.b Spouses or family members of CFS patients.
.sup.c Contacts are medical personnel coming in contact with CFS patients
Laboratory findings among CFS patients have shown low level autoantibodies
which may reflect an underlying autoimmune disease (Buchwald et al.,
Review Infect. Dis., 1991, 13:512-18). Evaluation of patients with SLE
showed only increased expression of the CD38 marker on CD8.sup.+ cells
(Table V); other cell surface markers were within the normal range.
Example 2
Isolation of Infectious Agent for CFS
1. Inoculation of Animals
One method for identifying a new agent involved in human disease is to
infect a susceptible animal with putatively diseased tissue. The animals
can be observed for the development of symptoms, and the agent can be
detected by standard microbiological and serological methods. We use
newborn NSF mice selected because of their low level endogenous virus
production (Levy Current Topics in Microbiology and Immunology (1978)
7:111), and the sensitivity of mice to a variety of human agents (Hsiung,
Diagnostic Virology (1982); and Prusiner, Ann. Rev. Micro. (1989) 43:345).
Syrian hamsters are chosen for their susceptibility to several infectious
agents including human parvoviruses and "prions," (Fraenkel-Conrat,
Virology (1988); Prusiner, Ann. Rev. Micro. (1989) 43:345; and Berns,
Plenum Press (1984)), their relative ease of study, and low cost of
purchase and maintenance. After inoculation with patient material, each
animal is observed for development of physical abnormalities or disease.
The animals are inoculated intraperitoneally (IP) or subcutaneously (SC)
with the following patient specimens:
a. 0.1-0.2 ml whole blood
b. separated PMC (10.sup.3-10.sup.6 cells) in 0.2 ml
Control animals receive blood and PMC from normal, healthy donors. In
addition, some hamsters receive material intracerebrally, since this
approach has helped detect the presence of human parvoviruses and prions
(Fraenkel-Conrat, Virology (1988); Prusiner, Ann. Rev. Micro. (1989)
43:345; and Berns, K.I. Plenum Press (1984).
All specimens are inoculated within 24 hrs. after removal from the subject.
The PMC of the CFS patient are separated by Ficoll/Hypaque density
gradient as is routinely conducted in the laboratory (Castro, J. Clin.
Microbiol. (1988) 26:2371). The PMC are suspended in RPMI 1640 medium
without serum for inoculation into the animals. Similar studies with small
animals were conducted in the initial attempts to find the AIDS virus,
(Morrow, J. Gen. Virol. (1987) 68:2253).
For these studies, the specimens from 10 CFS patients are injected into 100
newborn animals from each species each year. These animals are followed
for any signs of disease, including neurologic abnormalities or changes in
their immune responses (see below). Litter mates receive blood or PMC from
5 normal donors as controls.
2. Studies on Inoculated Animals
a. The animals are followed daily for any signs of disease. A full physical
examination is conducted weekly. They are evaluated for failure to thrive,
ruffled fur, enlargement of the spleen or thymus, pneumonia and neurologic
disorders (unsteady gait or paralysis).
b. For the first three months, control animals and those inoculated with
patient material are bled once every two weeks either intraorbitally or
from the tail vein. A complete blood count and differential is done. In
particular, activation markers on the CD8+ cells of mice (for which
markers are available) are measured (Eichmann, Immunol Rev. (1989)
109:39). In hamsters, the relative presence of lymphocytes, and
macrophages is assessed and compared to controls. Serum is collected
monthly to test for a potential agent that has hemagglutinating activity
against human, chick, goose, guinea pig and sheep erythrocytes as measured
by conventional techniques (White, Medical Virology (1986) and Hsiung,
Diagnostic Virology (1982)). These approaches were used in the initial
search for the AIDS virus. After three months, these studies are repeated
every 6 weeks or when animals show signs of disease. All animals are
maintained for one year.
c. Any sick animal or animal showing a change in normal immunologic profile
is sacrificed and evaluated using the following protocol: tissues are
examined for gross pathology and fixed for microscopic examination (see
Table A). Any tissues showing abnormalities are examined by electron
microscopy (EM) (see Table B). Extracts of the tissues and the blood are
reinoculated into newborn animals of the same strain in an attempt to
transfer the putative etiologic agent. They are also inoculated onto
selected cultured animal and human cells that are examined for evidence of
a pathogen such as CPE, hemadsorption, RT activity (see Table 6). With
some sick animals, attempts are made to establish the animal cells,
particularly PMC, in culture for further evaluation.
d. The PMC of the mouse strains separated by Ficoll/Hypaque procedures are
grown in the presence of mouse T cell growth factor (Smith, Immunol. Rev.
(1980) 51:337), which is available commercially. Human IL-2 also can be
used for these procedures as well as those using separated PMC from
hamsters. Supernatants from inoculated cells and cell lines are checked
for reverse transcriptase activity (Mn++ and Mg++ dependent) and
hemagglutinating activity (White, Medical Virology (1986) and Hsiung,
Diagnostic Virology (1982). If positive, they are inoculated back into
animals to assess the presence of a pathologic agent.
e. Sera from the animals, after absorption with normal human lymphocytes,
is tested by immunofluorescence assays (IFA) for reactivity with PMC
cultures of individuals with CFS and with cells inoculated with specimens
from these animals. Any positive result may reflect growth of the agent in
the animal host.
3. Tissue Culture Studies
a. Whole blood, PMC, sera, saliva and cerebrospinal fluid specimens from
patients with CFS are evaluated for the presence of an infectious agent by
inoculation onto normal PMC cultures and a variety of cell lines (see
Table VI), following pretreatment with polycations to increase their
sensitivity to virus infection (Castro, J. Clin. Microbiol. (1988)
26:2371).
TABLE VI
______________________________________
Cell Lines Used for Detection of the CFS Agent*
Animal Cell Lines
Human Cells
______________________________________
mouse 3T3 Sup-T - T cell line
BHK hamster MT-4 - T cell line
chicken embryo U937 - monocyte cell line
mink lung RAMOS - B cell line
bat lung Raji - B cell line
NRK - (rat) kidney
HOS - human fibroblastoid
Vero (monkey) cells
human skin fibroblast
SK-N-Mc - neuroblastoma
cells
______________________________________
*Cell lines are routinely checked for mycoplasma contamination. These
lines are selected because of their cell type and use in the laboratory
for growth of known viruses.
These cells are subsequently examined for an infectious agent by the
procedures listed in Table VII, including hemadsorption procedures using
guinea pig and chick erythrocytes (White, Medical Virology (1986) and
Hsiung, Diagnostic Virology (1982)). This latter approach was instrumental
in initially finding the simian type D retrovirus (Marx, Science (1984)
223:1083).
TABLE VII
______________________________________
Procedures to be Used Routinely for Detection of an
Infectious Agent in Cultures of CFS Tissues
______________________________________
1. Cytopathic effects (balloon degeneration, enlarged cells,
syncytia)
2. Hemadsorption (cells)
3. Hemagglutination (fluid)
4. Reverse transcriptase activity (Mg.sup.++, Mn.sup.++) (fluid)
5. Induction of antigens in the cells (detected by IFA)
______________________________________
Supernatant from these cultures is passed twice to fresh cells of the same
type to enhance the sensitivity for detection of the agent. Endogenous
retroviruses can be expressed in some of the cell lines (e.g., mouse, rat,
mink, MT-4) (Levy, Cancer Res. (1986) 46:5457 and Levy, Current Topics in
Microbiol. and Immunol. (1978) 7:111) and any RT activity is assessed for
its association with an endogenous virus. Specific antisera against these
agents are available.
4. Isolation of an infectious agent
Our laboratory, over the past four years, has evaluated peripheral blood
mononuclear cells (PMC), cerebrospinal fluid (CSF), saliva, and cervical
swabs obtained from 96 patients with CFS for the presence of an infectious
agent (see Table VIII).
TABLE VIII
______________________________________
Tissue Culture Studies to Detect Infectious Agents in
Chronic Fatigue Syndrome
Specimen No. Tested
No. Positive#
______________________________________
PMC 96 8
Plasma 24 1
CSF 2 0
Saliva 15 1
Cervical swab 1 0
______________________________________
#See Table 4 for details on positive specimens.
These studies involved the culturing of the PMC with and without
mitogen-stimulated normal PMC similar to approaches used to isolate the
AIDS virus (Castro, et al., J. Clin. Microbiol. (1988) 26:2371). In
addition, in some studies, the isolation of a potential agent was
attempted on a variety of different animal and human cell lines (see Table
VII). Thus far, evidence of a potential cytopathic agent has been noted in
eight of the individuals studied (Table VIII). Cytopathology was observed
in the PMC cultures of three, and in three others when fluid from the PMC
culture was passed to the RAMOS B cell line (see Table IX).
TABLE IX
______________________________________
Detection of Infectious Agents Associated with Chronic
Fatigue Syndrome
Specimens Cells Detected
How Detected
______________________________________
PMC PMC CPE
PMC PMC CPE
PMC PMC CPE
PMC PMC RT
PMC PMC RT
PMC PMC RT
PMC Ramos CPE
PMC Ramos CPE
PMC Ramos CPE
Saliva* PMC CPE
Plasma* PMC CPE
______________________________________
RT Mn++-dependent reverse transcriptase activity in the culture fluid.
CPE cytopathic effects as demonstrated by giant cells and balloon
degeneration in the cells listed (FIG. 1).
PMC peripheral blood mononuclear cells.
*PMC of patient also gave evidence of an infectious agent.
In 1 out of 15 patients, saliva induced cytopathic changes in the PMC
culture. Neither of 2 cerebrospinal fluids nor a single cervical specimen
showed any CPE-inducing agent, but one out of fifteen plasma samples gave
CPE in RAMOS cells. The observations with saliva were most likely due to
HHV-6 as this virus has recently been found in the saliva of most
individuals (Levy, et al., The Lancet (1990) 335:1047). None of the animal
or other human cells showed evidence of any agent by morphologic or
serologic criteria (e.g., IFA).
As noted above, in six PMC cultures an agent was found that led to
cytopathic changes in the RAMOS B cell line. Subsequent studies using
immunofluorescent techniques (conducted by E. Lennette) indicated that
this CPE was associated with staining of 0.1% of the cells by CFS sera;
the results did not reflect infection by EBV, HHV-6 or any other human
virus considered (see Table X for list).
TABLE X
______________________________________
Antibodies to Human Viruses in CFS Patients and Controls
CFS Control
Viral Agent Positive Positive
______________________________________
Adenovirus type 2
N = 64 19 (30%) N = 10 5 (50%)
Coxsackie B4
N = 64 35 (55%) N = 10 6 (60%)
N = 64
CMV N = 92 45 (49%) N = 10 5 (50%)
Measles N = 28 26 (89%) N = 28 26 (92%)
EBV N = 64 63 (89%) N = 10 9 (90%)
HHV-6 N = 52 51 (98%) N = 59 58 (99%)
HIV N = 96 0 (0%) N = 59 0 (0%)
HTLV-I/II N = 64 0 (0%) N = 10 0 (0%)
______________________________________
Subsequent passage of supernatant from the cultures to fresh RAMOS cells
did not lead to the isolation of an infectious agent. The induction of CPE
and antigen was achieved for 2-3 passages and then these cell culture
observations were no longer observed. In the final cultures in which the
virus was not noted, attempts to activate the agent with halogenated
pyrimidines (e.g., IUDR) also did not recover a CPE-inducing agent.
The fluid from one PMC culture induced cytopathic changes leading to cell
death in macrophage cultures from normal controls (Table VIII). Passage of
this filterable CPE-producing agent in the supernatant was achieved for 2
passages and then lost as was our experience with the RAMOS cell agents
described above.
Finally, in 31 PMC cultures evaluated for reverse transcriptase (RT)
activity (Hoffman, et al., Virology (1985) 147:326), 3 cultures gave
levels >10,000 cpm/ml of culture supernatant using Mn.sup.++ as the
cation. No RT activity with Mg.sup.++ was noted. For these studies, we
routinely used frozen and thawed cell pellets to detect any
cell-associated viruses as is the case with HTLV-1 (Fraenkel-Conrat, et
al., Virology 2nd Ed. (1988); and Levy, Cancer Res. (1986) 46:5457).
However passage of the RT activity to fresh cultures did not lead to
induction of RT activity nor isolation of a retrovirus. We have presumed
the initial RT activity reflected cellular polymerases.
b. PMC from CFS patients is cultured in RPMI 1640 medium with and without
IL-2, with and without PHA, and cocultivated with PMC from normal donors
(obtained from Irwin Memorial Blood Bank, San Francisco) or the Ramos cell
line. (Castro, J. Clin. Microbiol. (1988) 26:2371). These approaches
permit relative preferential growth of white cell subsets that may or may
not yield the CFS agent. (Castro, J. Clin. Microbiol. (1988) 26:2371). The
Ramos line is used because of its previous signs of CPE after inoculation
with PMC cell fluid from a CFS patient (see FIG. 1). These cultures are
examined for CPE and RT activity, and other signs of an infectious agent
(see Table 6) including induction of antigens detected by IFA using the
patient's serum. Fluid is also inoculated onto selected animal and human
cell lines (see Table VI) for further evaluation of a replicating agent
(see FIG. 2 for summary of this approach).
c. Sera from individual patients is used to examine the cells by IFA for a
potential infectious agent.
d. Sera from individual patients is also examined for the presence of an
infectious agent by measuring hemagglutinating activity using conventional
assays with sheep, chick, guinea pig, and human erythrocytes (White,
Medical Virology (1986)).
e. Culture of Purified subsets of peripheral white cells obtained from CFS
patients
1) Attempts will be made to identify the CFS infectious agent in culture by
techniques developed for efficient isolation of the AIDS virus. In
particular, selective culture of CD8+, CD4+, T-cells, B-cells, macrophages
and NK cells is conducted to examine the possible presence of the CFS
agent in one of these cell types. The cells are cultured selectively since
one cell type may prevent replication of the agent in another cell type.
For example, removal of CD8+ cells from some PMC cultures has enabled
recovery of HIV from the cells when the virus could not be detected
otherwise (Walker, Science (1986) 234:1563 and Immunology (1989) 66:628).
The cell selection and depletion is undertaken using immunomagnetic (IM)
beads (Gaudemack, et al., J. Immunological Methods (1986) 90:179). The PMC
are isolated from each sample using monoclonal antibody-coated beads
(Dynabeads, M-450 Dynal, Oslo, Norway) with selectivity for the major
human cell subsets. Twenty to fifty million PMC are suspended in 4-5 ml of
phosphate buffered saline (PBS) containing 2% heat-inactivated (30 min.,
56.degree. C.) fetal calf serum (FCS). This single cell suspension is
added to the IM beads (previously washed 3 times with cold PBS containing
2% FCS) to yield a bead:target cell ratio of approximately 3:1. The bead
cell suspension is incubated at 4.degree. C. on a rotating rocking machine
for 30-35 min. Cells rosetting with IM beads are captured by placing the
tube in a magnetic device (Dyno magnetic particle concentrator) for 1-2
min. followed by removal of the supernatant containing the non-bound
cells. The rosetted cells are washed 3 times with cold PBS containing 2%
FCS using the magnetic capture device noted above and then counted. The
non-adherent cells are subsequently treated with other IM beads coated
with a different monoclonal antibody and the procedure repeated.
This approach with IM beads can also be performed by first reacting the PMC
with mouse monoclonal antibodies to specific cell surface markers. Then,
beads with antibodies to mouse immunoglobins are added to select or
deplete a specific cell type. The purity of the subset is analyzed by
double-staining procedures on a Becton-Dickinson FACscan using specific
monoclonal antibodies (Levy, et al., Clin. Immunol. Immunopathol., (1985)
35:328).
2). All cells obtained from the blood are cultured in the presence of IL-2
(Pharmacia, Silver Spring, Md.) except for macrophages. They are grown in
10% FCS and 5% human (AB+) serum as described in Homsy, et al., Science,
(1989) 244:1357. The cells are examined for modulation of cell surface
markers by FACS analysis (see below) and for morphologic changes. The
cells and fluids are monitored for an infectious agent every 3 days, for
up to 28 days by the procedures listed in Appendix 6. This period of time
has been found necessary for detection of HIV in the cells of some
infected individuals (Castro, et al., J. Clin. Microbiol., (1985)
26:2371). The cells are activated with PHA, pokeweed mitogen (PWM), or
another mitogen specific for each cell type. Supernatants from the
cultures are obtained and passed to fresh cells from normal controls.
Included in these studies is the cultivation of the different cells or
their fluids with established human T, B, and macrophage cell lines, and
animal cell lines (see Appendix 3). Induction of CPE, RT activity, or
specific IFA staining would be used to detect infection. Finally, to
augment detection of an agent, halogenated pyrimidines (e.g., IUDR) to
activate virus replication are used.
f. Specific Studies of Macrophages
In the initial studies (see Preliminary Studies, Section C) CPE in normal
macrophages inoculated with supernatant fluid from the PMC culture of an
individual with CFS was found. To rule out a cytokine effect, filtered
material from these cultures was then passed to fresh macrophages and CPE
was again noted as demonstrated by cell death and granular formation in
the cells. However, continued passage of this CPE-inducing effect was not
achieved.
These studies are being repeated using macrophages of CFS patients as a
source of supernatant fluid. They are obtained by adherence to plastic as
described in Homsy, et al., Science, (1989) 244:1357. Trypsinization is
used to maintain purity of the cells. Cells are followed for the presence
of an infectious agent as described (see Appendix 6). In addition, fluids
from the macrophages as well as CDS+, CD4+, B cells and NK cells from CFS
patients are inoculated onto macrophages from control individuals to
evaluate whether there is a transferable morphologic effect on these cells
in culture.
The culture supernatants from CFS macrophages are also inoculated onto a
selection of animal and human cells (see Appendix 3) and their possible
induction of CPE, RT activity, and antigen is evaluated as noted above.
g. Specific Studies with NK Cells
A common finding in CFS is decreased NK cell function (Kibler, et al., J.
Clin. Immunol. (1985) 5:46; Murdoch, NZ Med J (1986) 1015511; Caligiuri,
et al., J. Immunol (1987) 139 (10):3306; Klimas, et al., J. Clin. Micro.
(1990) 28(6):1403). Therefore, a patient's NK cells purified by CD16/CD56
IM beads, or the cell culture fluid is cocultured with normal NK cells to
transfer an infectious agent detected as described (see Table 6). These
normal NK cells are assessed for function by measuring their ability to
kill .sup.51 CR-labelled K562 cells (Baron, et al., Diagn. Immunol. (1985)
3:197; Hudson, et al., Practical Immunology, 2nd Ed. Blackwell Scientific
Publications, Oxford (1980), (see Section C)).
h. Specific Studies with CD8+ Cells
We have found that the CD11b+ cells (suppressor CD8+ cells) decrease during
CFS. We plan to select for the CD8+/CD11b+ cells with IM beads and examine
them for a possible infectious agent. The procedures are similar to those
described above for NK cells. In addition, supernatant from the CD11b+
cells is removed and added to CD11b+ cells of normal donors to see if any
cytopathic effects are noted that could explain the reduction in this
particular cell subset. An effect on expression of cell surface markers is
measured by FACS analysis. Cells are monitored for an infectious agent as
described in Table 6. In addition, an effect of the cell supernatant from
CD11b+ cells on mitogen-induced proliferation of normal lymphocytes and
their production of gamma interferon is evaluated (Klimas, et al., J.
Clin. Micro. (1990) 28(6):1403; Hudson, et al., Practical Immunology, 2nd
Ed. Blackwell Scientific Publications, Oxford (1980). (see Section C)).
4. PCR Analysis
To evaluate whether an occult retrovirus might be the cause of CFS, PCR
analysis is performed on PMC specimens from CFS patients and controls with
gag and polymerase probes of HTLV-1, HTLV-II and HIV. In brief;
2.times.10.sup.6 cells are extracted and the PCR performed on the DNA as
described in Ou, C--Y, et al., Science (1988) 239:295, Innis, PCR
Protocols, Academic Press (1990). Two separate gag specific primer base
pairs for HIV are used: SK38/39 and SK101/145. A set of gag primers,
SG166/SK296, and a set of pol primers, SK110/111, for HTLV-I is used. The
Cetus thermocycler is used for 30 cycles each for 30 sec at 95.degree.,
55.degree. and 72.degree. C. respectively. The amplified product is
hybridized to oligomer probes for HIV-1 (SK19 and SK102) and probes to
HTLV-I gag (SG242) and pol (SK112, 1SK18). The resulting products are
analyzed by polyacrylamide gel electrophoresis and autoradiography.
Similar PCR evaluation is performed using DNA probes characteristic for
other viral families such as parvoviruses, herpes viruses, papilloma
viruses, and enteroviruses.
All publications and patent applications cited in this specification are
herein incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of
ordinary skill in the art that many changes and modifications can be made
thereto without departing from the spirit or scope of the appended claims.
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